1. Field of the Invention
This invention relates to the administration of proteins to living skin cells. In connection with some of its specific aspects, it relates to DNA repair enzymes and, in particular, to 1) methods for purifying DNA repair enzymes, and 2) methods and means for administering DNA repair enzymes to living cells in situ, e.g. human skin cells, so that the enzymes can enter the cells and enhance the repair of damaged DNA in the cells.
2. Description of the Prior Art
Skin cancer is a serious human health problem. The incidence of non-melanoma skin cancer in the United States is 500,000 per year, and 23,000 per year for melanoma. Annual deaths are 2,000 and 6,000 respectively, and 800,000 deaths from skin cancer are predicted in the next 88 years if current trends continue.
The casual link between non-melanoma skin cancer and ultraviolet light exposure from the sun has been clearly established, and sun exposure is an important causative factor in melanoma. The target for ultraviolet light damage leading to cancer is widely acceptable as DNA.
Xeroderma pigmentosum is a human genetic disease in which patients develop solar damage, pigmentation abnormalities and malignancies in sun-exposed skin. A review of the disease was authored by J. H. Robbins, H. H. Kraemer, M. A Lutzner, B. W. Festoff and H. G. Coon, entitled "Xeroderma Pigmentosum: An Inherited Disease with Sun Sensitivity, Multiple Cutaneous Neoplasms, and Abnormal DNA Repair ", and published in the ANNALS OF INTERNAL MEDICINE, volume 80, number 2, pages 221-248, February, 1974. The disease occurs in 1 of 250,000 worldwide. Cells from xeroderma pigmentosum patients are deficient in repair of ultraviolet damage to DNA, which results in a cancer incidence 4,800 times the frequency of the general U.S. population. There is no cure, and treatment consists of avoiding sun exposure and excising skin lesions. Death occurs 30 years earlier in these patients than among the general U.S. population.
Research into the basic mechanisms of DNA repair has established outlines of biochemical pathways which remove ultraviolet damage in DNA. Bacterial repair systems have been demonstrated to differ significantly from repair inhuman cells. However, enzyme endonuclease V (also referred to herein as T4 endonuclease V and denV endonuclease V) has the ability to enhance DNA repair in human cells as evidenced by increased UV-specific incision of cellular DNA, increased DNA repair replication, and increased UV survival after treatment with the enzyme.
The endonuclease V enzyme is produced by the denV gene of the bacteriophage T4. It has been established that this enzyme catalyzes the rate limiting, first step in the removal of UV-induced DNA damage, namely, single strand incision of DNA at the site of damage. In particular, the enzyme exhibits glycosylase and apurinic/apyrimidinic endonuclease activities and acts at the site of ultraviolet induced pyrimidine dimers. See "Evidence that the UV Endonuclease Activity Induced by Bacteriophage T4 Contains Both Pyrimidine Dimer-DNA Glycosylase and Apyrimidinic/Apurinic Endonuclease Activities in the Enzyme Molecule" by H. R. Warner, L. M. Christensen and M. L. Perrsson, in JOURNAL OF VIROLOGY, 1981, Vol. 40, pages 204-210; "denV Gene of Bacteriophage T4 Codes for Both Pyrimidine Dimer DNA Glycosylase and Apyrimidinic Endonuclease Activities" by S. McMillan, H. J. Edenberg, E. H. Radany, R. C. Friedberg and E. C. Friedberg, in JOURNAL OF VIROLOGY, 1981, Vol 40, pages 211-223, and "Physical Association of Pyrimidine Dimer DNA Glycosylase and Apurinic/Apyrimidinic DNA Endonuclease Essential for Repair of Ultraviolet-damaged DNA" by . Nakabeppu and M. Sekiguchi, in PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, 1981, Vol. 78, pages 2742-2746.
Other enzymes having the ability to repair DNA damage have also been identified. These enzymes include O.sup.6 -methylguanine-DNA methyltransferases, photolyases, uracil- and hypoxanthine-DNA glycosylases, apyrimidinic/apurinic endonucleases, DNA exonucleases, damaged-bases glycosylases (e.g., 3-methyladenine-DNA glycosylase), correndonucleases alone or in complexes (e.g., E. coli uvrA/uvrB/uvrC endonuclease complex), and other enzymes and enzyme complexes whose activities at present are only partially understood, such as, the products of the ERCC genes of humans and the RAD genes of yeast. Various of these enzymes have been purified to homogenity from microorganisms, and the genes for some of the enzymes have been cloned. As used herein, the term "DNA repair enzymes" is intended to include the foregoing enzymes, the T4 endonuclease V enzyme, and other enzymes now known or subsequently discovered or developed which have the ability to participate in repair of damaged nucleic acids and, in particular, damaged DNA.
To data, the use of exogenous enzymes in DNA repair systems has been limited to laboratory experiments designed to study the biochemical and evolutionary relationships among DNA repair pathways. Clinical application of these laboratory results has not been undertaken because, inter alia, there has been no effective way of purifying commercial quantities of DNA repair enzymes and there has been no effective, non-toxic way of administering DNA repair enzymes to living cells. The present invention addresses both of these long-standing problems in the art.
Purification of DNA enzymes for commercial use requires a homogenous final product, high yield, speed, simplicity and low cost. The existing methods of the art have been unable to meet these goals, as follows:
(1) P. Seawell, E. C. Friedberg, A. K. Ganesan and P. C. Hanawalt, "Purification of Endonuclease V of Bacteriophage T4" in DNA REPAIR; A LABORATORY MANUAL OF RESEARCH PROCEDURES, edited by E. C. Friedberg and P. C. Hanawalt, Marcel Dekker, Inc., New York, 1981, Volume 1, Part A, pages 229-236.
This method uses phage T4 infected E. coli, and purification relies on phase-separation and two ion-exchange chromatography steps (DEAE- and phospho-cellulose). The DEAE chromatography step limits the yield of the method because all proteins must bind in order to elute the enzyme of interest. The method is not rapid; each chromatography step is preceded by dialysis, each elution requires at least 20 hours, and each fraction is assayed for activity. The process is neither simple nor inexpensive: tedious phase separation and repetitive assays are performed, and all spent dialysate and separated phases are discarded. Significantly, the authors of this method describe their final product as being only partially purified.
The basic steps of the Seawell et al. method were first described by E. C. Friedberg and J. J. King in "Dark Repair of Ultraviolet-irradiated Deoxyribonucleic acid by Bacteriophase T4: Purification and Characterization of a Dimer-Specific Phage-Induced Endonuclease", JOURNAL OF BACTERIOLOGY, 1971, Vol. 106, pages 500-507. This earlier version of the method included an additional DNA-cellulose step, which was omitted in the later version. A method similar to the Friedberg and King method was described by S. Yasuda and M Sekiguchi, "T4 Endonuclease Involved in Repair of DNA" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, December, 1970, Vol. 67, pages 1839-1845. Instead of using a DNA-cellulose step as in the Friedberg and King method, the Yasuda and Sekiguchi method included an optional gel filtration step.
(2) Y. Nakabeppu, K. Yamashita and M. Sekiguchi, "Purification and Characterization of Normal and Mutant Forms of T4 Endonuclease V" JOURNAL OF BIOLOGICAL CHEMISTRY, 1982, Vol. 257, pages 2556-2562.
The basic steps of this method were first described by S. Yasuda and M. Sekiguchi, "Further Purification and Characterization of T4 Endonuclease V", BIOCHIMICIA ET BIOPHYSICA ACTA, 1976, Vol. 442, pages 197-207. These methods are similar to the Seawell et al. method, except that they substitute cation exchange (carboxymethyl Sephadex) chromatography for anion exchange (DEA) chromatography, and add additional chromatography steps including either hydroxylapatite or gel filtration and UV DNA cellulose (the Yasuda and Sekiguchi method also differs from the Seawell et al. method in that it does not include a phosphocellulose step). These methods have the same difficulties as the Seawell et al. method, with the additional problems of lower yield, less speed and simplicity, and greater cost.
(3) K. M. Higgins and R. S. Lloyd, "Purification of the T4 EndonucleaseV", MUTATION RESEARCH, 1987, Vol. 183, pages 117-121.
This method uses as E. coli strain which harbors a plasmid containing the phage T4 denV structural gene under the control of the phage lambda rightward promoter. The chromatography steps are single-stranded DNA agarose, chromatofocusing and cation exchange (carboxymethyl-Sephadex). The yield is low compared to the present invention, in that 12 liters of bacteria are required for 15 mg pure enzyme. The yield is also limited by the requirement that all proteins bind to the chromatofocusing column in order to elute the desired enzyme. The method is not rapid: each chromatography step is preceded by dialysis and concentration by ultrafiltration; at least two of the steps require on the order of 17.5 hours for elution; and each step is followed both by enzyme activity assays and polyacrylamide gel analysis of each fraction. The method is not simple: the single-stranded DNA agarose chromatography requires pooling of 84% of the collected fraction (520 ml of 700 ml eluent), extensively diluting the loaded protein; experiments in connection with the present invention showed that the chromatofocusing step was not reproducible using DEAE agarose and Servalyte ampholines; ultrafiltration is required in addition to dialysis; and tedious, repetitive activity assays and gel analysis are performed after each step. The method is expensive: large ultrafiltration devices are used and discarded at every step; the single-stranded DNA agarose is exposed to crude bacterial lysates with active nucleases which drastically reduce the useful life of the column; and costly chromatofocusing reagents including Pharmacia PBE 94 gel and polybuffer ampholines must be used.
In addition to the foregoing, two methods have bene published for the purification of O.sup.6 -methylguanine-DNA methyltransferase. See B. Demple, A. Jacobsson, M. Olsson, P. Robbins and T. Lindahl, "Repair of Alkylated DNA in Escherichia coli: Physical properties of O.sup.6 -methylguanine-DNA methyltransferase" in THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 257, pages 13776-13780, 1982, and Y. Nakabeppu, H. Kondo, S. Kawabata, S. Iwanaga and M Sekiguchi, "Purification and Structure of the Intact Ada Regulatory Protein of Escherichia coli K12 O.sup.6 -Methylguanine-DNA Methyltransferase" in THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, pages 7281-7288, 1986. The Demple method uses phosphocellulose chromatography before DNA-cellulose and gel filtration, and includes a final phenylagarose chromatography step. The Nakabeppu method uses two rounds of ion-exchange (DEAE-) chromatography followed by phosphocellulose and gel filtration chromatography.
A general review of purification methods for DNA repair enzymes can be found in DNA REPAIR: A LABORATORY MANUAL OF RESEARCH PROCEDURES, edited by E. Friedberg and P. C. Hanawalt, published by Marcel Dekker, New York. Volume I, part A, of this text contains methods for purifying five enzymes: photolyase, endonuclease V (discussed above), AP endonuclease, uracil-DNA glycoxylase and hypoxanthine-DNA glycosylase, in chapters 18-22, respectively. Volume II, chapters 3-5, discuss the Demple method referred to above and methods for purifying 3-methyladenine-DNA glycosylases. Volume III, Section IV, contains methods for purification of photolyase, the uvrABC excision nuclease and the uvrD helicase in chapters 23-25. None of these methods, nor the two methods discussed above for purifying O.sup.6 -methylguanine-DNA methyltransferase, use of purification procedures of the present invention.
Various approaches have bene considered in the field of DNA repair for delivering DNA repair enzymes to mammalian cells. The goal of these efforts has been to discover and characterize the pathways of DNA repair in mammalian cells and their evolution, not to develop commercial methods for augmenting DNA repair. Thus, researches have not used normal cells, such as skin epidermal keratinocyte cells, as target cells, but rather have concentrated on fibroblasts from patients with xeroderma pigmentosum. Similarly, prior research has focused on non-physiological techniques for introducing DNA repair enzymes into cells which are useful only in the laboratory and which compromise the physiology of the target cells. The published reports regarding this work include:
(1) K. Tanaka, M. Sekiguchi and Y. Okada, "Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus)", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES U.S.A., 1975, Vol. 72, pages 4071-4075; and K. Tanaka, H. Hayakawa, M. Sekiguchi and Y. Okada, "Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES U.S.A., 1977, Vol. 74, pages 2958-2962.
In these two reports, fibroblasts derived from patients with xeroderma pigmentosum were treated with inactivated Sendai virus and endonuclease V after UV irradiation. Proteins on the coat of the Sendai virus rendered the cells permeable to endonuclease V. This treatment enhanced DNA repair replication and increased survival of the treated cells. This method of introducing the enzyme is not practical for commercial application because of the pathogenicity of the Sendai virus. Large external enzyme concentrations are also required. In its discussion section, the Tanaka et al. reference discusses approaches to the study of the evolution of macromolecular (i.e. DNA repair) systems in organisms and mentions liposome methods and erythrocyte ghost/HVJ methods as other methods for introducing macromolecules into cells. Significantly, Tanaka et al. ultimately conclude that the Sendai visus method is the most simple and applicable method in basic research for the introduction of rather small macromolecules of about 20,000 daltons, i.e., the T4 endonuclease V molecule.
(2) G. Ciarrocchi and S. Linn, "A cell-free assay measuring repair DNA synthesis inhuman fibroblasts", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES U.S.A., 1978, Vol. 75, pages 1887-1891.
In this report, human normal and xeroderma pigmentosum fibroblasts were disrupted by osmotic shock after UV irradiation, and incubated with endonuclease V. DNA repair synthesis was increased in both types of cells, and repair in xeroderma pigmentosum cells increased to the level of normal cells. This method for introducing enzyme into cells was only employed for in vitro research, as it destroys the integrity of the cell membrane and viability is drastically affected. Large external enzyme concentrations are also required.
(3) D. Yarosh and R. Setlow, "Permeabilization of Ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus", MOLECULAR AND CELLULAR BIOLOGY, 1981, Volume 1, pages 237-244.
In this method, hamster cells were treated with polyethylene glycol after UV irradiation and then incubated with a DNA repair enzyme which acts similarly to endonuclease V. The enzyme entered the cells and acted on resident DNA. The method was toxic to target cells, probably because it relied on permeabilization, and vital molecules exited as the enzyme entered. This method also requires large external enzyme concentrations for efficacy.
(4) J. H. J. Hoeijmakers, "Characterization of genes and proteins involved in excision repair of human cells", JOURNAL OF CELL SCIENCE SUPPL., 1987, Vol. 6, pages 111-125.
This reference summarizes a body of research in which proteins were introduced into the nuclei of cells by microinjection. When endonuclease V was injected into the nuclei of xeroderma pigmentosum cells, DNA repair synthesis was increased. This method is applicable only for laboratory research.
(5) K. Valerie, A. P. Green, J. K. de Riel and E. E. Henderson, "Transient and stable complementation of ultraviolet repair in xeroderma pigmentosum cells by the denV gene and bacteriophage T4", CANCER RESEARCH, 1987, Vol. 47, pages 2967-2971.
In this method, the denV gene under the control of a mammalian promoter was tranfected into xeroderma pigmentosum cells. Clones selected for uptake of the denV gene showed increased incision of UV-DNA, enhanced DNA repair synthesis and increased resistance to ultraviolet irradiation. The transfection process is very inefficient (less than one success per million cells) for normal human cells. These methods fall into the category of gene therapy, and are beyond the scope of the current art for commercial use.
In a few laboratories, liposomes have been used in the topical delivery of drugs, but not of enzymes and, in particular, not of DNA repair enzymes. The reports of encapsulation and topical drug delivery include:
Delivery of triaminolone: Michael Mezei and Vijeyalkshmi Gulasekharam, "Liposomes--A selective drug delivery system for the topical route of administration, I. lotion dosage form", in LIFE SCIENCES, volume 26, pages 1473-1477, 1980; Michael Mezei and Vijeyalakshmi Gulasekharam, "Liposomes--A selective drug delivery system for the topical route of administration: gel dosage form", in JOURNAL OF PHARMACY AND PHARMACOLOGY, volume 34, pages 473-474, 1981. Delivery of tetracaine: Adrienn Gesztes and Michael Mezei, "Topical anaesthesia of the skin by liposome-encapsulated tetracaine", ANESTHESIA AND ANALGESIA, volume 67, pages 1079-1081, 1988. Delivery of methotrexate: H. M. Patel "Liposomes as a controlled-release system", in BIOCHEMICAL SOCIETY TRANSACTIONS, volume 13, pages 513-516, 1985. Delivery of hyrocortisone: W. Wohlrab and J. Lasch, "Penetration kinetics of liposomal hydrocortisone in human skin", in DERMATOLOGICIA, volume 174, pages 18-22, 1987.
The use of pH sensitive liposomes to mediate the cytoplasmic delivery of calcein and FITC dextran has been described in the following references: Robert Straubinger, Keelung Hong, Daniel Friend and Demetrios Papahadjopoulos, "Endocytosis of Liposomes and Intracellular Fate of Encapsulated Molecules: Encounter with a Low pH Compartment after Internalization in Coated Vesicles," CELL, volume 32, pages 1069-1079, 1983; and Robert Straubinger, Nejat Duzgunes and Demetrios Papahadjopoulos, "pH-Sensitive Liposomes Mediate Cytoplasmic Delivery of Encapsulated Macromolecules," FEBS LETTERS, volume 179, pages 148-154, 1985. Other discussions of pH sensitive liposomes can be found in chapter 11 of the book CELL FUSION, edited by A. E. Sowers, entitled "Fusion of Phospholipid Vesicles Induced by Divalent Catins and Protons" by Nejat Duzgunes, Keelung Hong, Patricia Baldwin, Joe Bentz, Shlomo Nir and Demetrios Papahadjopoulos, published by Plenum Press, N.Y., 1987, pages 241-267. See also Ellens, Bentz and Szoka, "pH-Induced destablization of phosphatidylethanolamine-containing liposomes: role of bilayer contact," BIOCHEMISTRY, volume 23, pages 1532-1538, 1984, and Bentz, Ellens and Szonka, "Destabilization of Phosphatidylethanolamine-Containing Liposomes: Hexagonal Phase and Asymmetric Membranes", BIOCHEMISTRY, volume 26, pages 2105-2116, 1987. None of these references discusses or suggests the use of pH sensitive liposomes to topically administer DNA repair enzymes to human skin.